Antigenic variation was observed in the expression of specific tumor associated antigens within individual human mammary tumor masses using monoclonal antibodies. This variation was demonstrated by both the pattern and cellular localization of reactivity with a given antibody. This diversity was also observed in human mamary tumor cell lines grown in vivo and in vitro. Analyses of DNA content and cell surface binding of monoclonal antibodies during logarithmic growth phase, and at density-dependent arrest, demonstrated that the expression of some tumor associated antigens is related to S-phase of the cell cycle. Membrane expression of the reactive antigens appeared to be stable despite prolonged exposure to antibody. Antigenic drift was observed with continued passage of mammary tumor cell lines; consistent with this finding, the "same" mammary tumor cell line obtained different sources exhibited distinct antigenic phenotypes. Single-cell clones derived from the MCF-7 mammary tumor cell line exhibited at least four distinct antigenic phenotypes; a change in cell surface phenotype of some of the clones was seen during subsequent passage. Antigenic modulation as well as heterogeneity was also observed for the expression of the antigen reactive with monoclonal B72.3. Monoclonal antibody B72.3 reacts with the majority of breast and colon carcinomas obtained from biopsy but with only 1/28 breast carcinoma cell lines and 2/19 colon carcinoma cell lines. Growth of one of the positive colon carcinoma cell lines, LS-174T, as tumors in athymic mice, caused a 100-fold increase in the antigen reactive with B72.3 in cell extracts and a 10-fold increase on the cell surface. Consistent with this finding, increased expression of the antigen reactive with B72.3 was observed when LS-174T cells were grown under culture conditions that promote three-dimensional growth of the cells.